Modelos de regressão aleatória para características de crescimento de bovinos da raça Nelore do estado de Mato Grosso / Random regression models to traits of growth in the Nellore breed in Mato Grosso state

Foram utilizados 138.976 registros de informações de pesos corporais variando de 60 a 610 dias de idade, provenientes de 27.327 animais da raça Nelore, oriundos de rebanhos do estado do Mato Grosso, com o objetivo de descrever a variabilidade genética e estimar parâmetros genéticos para o peso corporal em diferentes idades, utilizando-se modelos de regressão aleatória. O modelo empregado incluiu efeitos fixos de grupo de contemporâneos e idade da vaca ao parto como covariáveis, além de efeitos aleatórios genético aditivo direto, genético materno, ambiente permanente de animal, ambiente permanente materno e efeito de ambiente temporário. O modelo de regressão aleatória mais adequado foi o que empregou função de covariância com polinômios de quarta ordem para descrição da variabilidade de todos os efeitos e duas classes de variância residual. As estimativas de variância genética aditiva direta e de ambiente permanente de animal aumentaram com a idade dos animais. As variâncias genética materna e de ambiente permanente materno exibiram comportamento semelhante, com maiores valores na fase de aleitamento. Os coeficientes de herdabilidade estimados variam de 0,25 a 0,43, com maiores valores nas idades mais avançadas na trajetória de crescimento dos animais. Esses resultados indicaram presença de variabilidade genética suficiente para obtenção de ganho genético expressivo por meio da seleção, principalmente após desmama. Os resultados encontrados para a correlação genética aditiva direta exibiram baixas correlações entre pesos nas idades iniciais e finais, porém pesos altamente correlacionados entre idades mais próximas. As correlações genéticas estimadas entre os pesos da desmama com os pesos até 610 dias de idade foram altas e positivas e indicam que os genes responsáveis por maiores pesos nesse período, em sua maioria, são os mesmos.

In this study 138,976 records of live weight between 60 to 610 days of age, from 27,327 Nellore cattle breed, from herds in Mato Grosso State were used in order to describe the genetic variability and to estimate genetic parameters for the live weight at different ages, using random regression models. The model included the fixed effects of contemporary group and age of cow at calving as covariate, random effects of direct additive genetic, maternal genetic, animal and maternal permanent environmental and temporary environment effect. The most appropriate random regression model employed the covariance function with fourth order polynomials to describe the variability of all effects and two residual variance classes. Estimates of direct additive genetic variance and animal permanent environment increased with the age of the animals. Maternal genetic variances and maternal permanent environment exhibited similar behavior, with higher values in pre weaning. The estimated heritability coefficients ranged from 0.25 to 0.43, with higher values at older ages in the growth trajectory of the animals. These results showed the presence of sufficient genetic variability to obtain significant genetic gain through selection, especially after weaning. The results for the direct additive genetic correlation exhibited low correlations between weights in initial and final ages, however, highly correlated weights between nearest ages. Genetic correlation estimates between weaning with weights up to 610 days of age were high and positive and indicate that most of the genes responsible for higher weights in this period are the same.

Transduction of CD34+ cells with lentiviral vectors enables the production of large quantities of transgene-expressing immature and mature dendritic cells.

BACKGROUND: Genetically engineered dendritic cells (DC) presenting specific antigens to T cells may be of great interest for immunotherapy. For this reason, the production of transgene-expressing DC derived from CD34 + cells transduced either shortly after ex vivo purification or during their differentiation into DC were evaluated. METHODS: CD34+ cells were transduced with lentivectors encoding for GFP before or after 21 days of culture with FLT3-ligand, thrombopoietin and stem cell factor and induction into DC with GM-CSF+IL-4 (G4) or G4+TNF (GT4). GFP and DC-specific marker expression was assessed by flow cytometry, and allostimulatory capacity was evaluated on GFP+ and GFP- sorted cells. RESULTS: Immature (G4-induced) DC obtained from amplified CD34 + cells were transducible by lentiviral vectors while mature (GT4-induced) DC were rather refractory. Moreover, since differentiated DC did not proliferate, large quantities of vectors were required to generate transgene-expressing cells with this protocol. In contrast, greater numbers of both immature and mature GFP- expressing DC were obtained with CD34+ cells exposed to lentivector shortly after purification. By the time of DC induction, GFP+ cells had increased by approximately 170-fold. After DC induction with G4, 32% of CD1a+, HLA-DR+, or CD40+ cells expressed GFP. CD1a+E-cadherin+ GFP+ Langerhans-like DC were also obtained. Incubation with TNF induced mature CD83+GFP+ DC that displayed a higher allostimulatory capacity than cells induced with G4 alone. CONCLUSION: The transduction of a small number of CD34+ cells with minimal doses of lentivector may allow for the production of a large number of DC expressing selected antigens useful for immunotherapy.

CD34(+) cord blood cells expressing cutaneous lymphocyte-associated antigen are enriched in granulocyte-macrophage progenitors and support extensive amplification of dendritic cell progenitors.

OBJECTIVE: We evaluated the frequency of hematopoietic progenitor cells (HPC) in CD34(+)CLA(+) (cutaneous lymphocyte-associated antigen) and CD34(+)CLA(-) cord blood cells, and followed cellular growth and HPC production during cultures in Flt3 ligand, thrombopoietin, and stem cell factor (FTS). MATERIALS AND METHODS: Immunomagnetic bead-purified CD34(+) cells were sorted into CD34(+)CLA(+) or CD34(+)CLA(-) cells. HPC frequency was assessed by clonal assays in methylcellulose either ex vivo or after, 7, 14, or 21 days of culture with FTS. Dendritic cell (DC) progenitors were evaluated after induction of FTS-amplified cells into DC using secondary cultures containing granulocyte-macrophage colony-stimulating factor and interleukin-4. RESULTS: Ex vivo, granulocyte-macrophage progenitors were more frequent and erythroid progenitors were less frequent in the CLA(+) fraction. In FTS culture, CD34(+)CLA(+) cells produced greater absolute numbers of CD34(+) cells, granulocyte-macrophage-, erythroid-, and DC (including Langerhans cell-related) progenitors compared to CD34(+)CLA(-) cells. In CD34(+)CLA(+) cultures, CLA(+) cells steadily decreased with time, and CD34(+)CLA(-) cells appeared. In CD34(+)CLA(-) cultures, CLA(+) cells were generated, increased up to day 7, and decreased thereafter. CLA was expressed only on CD34(-) cells in these cultures. Ex vivo, CD34(+)CLA(+) cells could be subdivided further into CD38(low) and CD38(high) cells. Cord blood and growth factor-mobilized CD34(+) cells contained more CLA(+)CD38(low) cells than nonmobilized peripheral blood CD34(+) cells and proliferated more extensively with FTS than the latter cells. CONCLUSIONS: CD34(+)CLA(+) cells contain a rather immature progenitor capable of high proliferation and extensive amplification of HPC in vitro. This progenitor may be localized in the CD34(+)CLA(+)CD38(low) fraction. In addition, cultures of CD34(+)CLA(+) cells from cord blood produced CD34(+)CLA(-) cells, suggesting that these cells may derive directly from CD34(+)CLA(+) cells in vivo.

IgG subclass switch capacity is low in switched and in IgM-only, but high in IgD+IgM+, post-germinal center (CD27+) human B cells.

Recent studies have shown that in humans the germinal center reactions produce three types of V(D)J mutated B cells in similar proportions, i.e. Ig-switched, IgD-IgM+ (IgM-only) and IgD+IgM+ cells, and that together they form the CD27+ compartment of recirculating B cells. We investigated the Ig isotype switch capacity of these cells. Peripheral blood B subsets were sorted and IgG subclass secretion in presence or absence of IL-4 was compared in B cell assays which lead to Ig secretion in all (coculture with EL-4 thymoma cells) or only in CD27+ (CD40L stimulation) B cells. Already switched IgG+ B cells showed no significant sequential switch and IgM-only cells also had a low switch capacity, but IgD+CD27+ switched as much as IgD+CD27- B cells to all IgG subclasses. Thus, in switched B cells some alterations compromising further switch options occur frequently; IgM-only cells may result from aborted switch. However, IgD+CD27+ human B cells, extensively V(D)J mutated and "naive" regarding switch, build up a repertoire of B cells combining (1) novel cross-reactive specificities, (2) increased differentiation capacity (including after T-independent stimulation by Staphylococcus aureus Cowan I) and (3) the capacity to produce appropriate isotypes when they respond to novel pathogens.

Naive and memory B cells in T-cell-dependent and T-independent responses.

This review focuses on the properties and roles of distinct subsets among the primary and the memory B lymphocytes regarding their contribution to helper-T-cell-dependent and -independent antibody responses. The naive/memory B cell functions are explained in the context of current concepts on the basic mechanisms of humoral immunity. Differences between murine and human B cells are also discussed.

Fas engagement induces the maturation of dendritic cells (DCs), the release of interleukin (IL)-1beta, and the production of interferon gamma in the absence of IL-12 during DC-T cell cognate interaction: a new role for Fas ligand in inflammatory responses.

Ligation of the Fas (CD95) receptor leads to an apoptotic death signal in T cells, B cells, and macrophages. However, human CD34(+)-derived dendritic cells (DCs) and mouse DCs, regardless of their maturation state, are not susceptible to Fas-induced cell death. This resistance correlates with the constitutive expression of the Fas-associated death domain-like IL-1beta-converting enzyme (FLICE)-inhibitory protein (FLIP) ligand. We demonstrate a new role of Fas in DC physiology. Engagement of Fas on immature DCs by Fas ligand (FasL) or by anti-Fas antibodies induces the phenotypical and functional maturation of primary DCs. Fas-activated DCs upregulate the expression of the major histocompatibility complex class II, B7, and DC-lysosome-associated membrane protein (DC-LAMP) molecules and secrete proinflammatory cytokines, in particular interleukin (IL)-1beta and tumor necrosis factor alpha. Mature DCs, if exposed to FasL, produce even higher amounts of IL-1beta. Importantly, it is possible to reduce the production of IL-1beta and interferon (IFN)-gamma during DC-T cell interaction by blocking the coupling of Fas-FasL with a Fas competitor. Finally, during cognate DC-T cell recognition, IL-12 (p70) could not be detected at early or late time points, indicating that Fas-induced, IFN-gamma secretion is independent of IL-12.

High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors.

Recent experiments point to the great value of lentiviral vectors for the transduction of human hematopoietic stem cells (hHSCs). Vectors used so far, however, have been poorly satisfying in terms of either biosafety or efficiency of transgene expression. Herein is described the results obtained with human immunodeficiency virus-based vectors optimized in both of these aspects. It is thus shown that vectors containing the EF1alpha and, to a lesser extent, the phosphoglycerate kinase (PGK) promoter, govern high-level gene expression in human hematopoietic progenitors as well as derived hematopoietic lineages of therapeutic relevance, such as erythrocytes, granulocytes, monocytes, dendritic cells, and megakaryocytes. EF1alpha promoter-containing lentiviral vectors can also induce strong transgene expression in primary T lymphocytes isolated from peripheral blood. A self-inactivating design did not affect the performance of EF1alpha promoter-based vectors but significantly reduced expression from the PGK promoter. This negative effect could nevertheless be largely rescued by inserting the post-transcriptional regulatory element of woodchuck hepatitis virus upstream of the vector 3' long terminal repeat. These results have important practical implications for the genetic treatment of lymphohematologic disorders as well as for the study of hematopoiesis via the lentivector-mediated modification of hHSCs.

BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth.

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family, designated BAFF (for B cell activating factor belonging to the TNF family), which is expressed by T cells and dendritic cells. Human BAFF was mapped to chromosome 13q32-34. Membrane-bound BAFF was processed and secreted through the action of a protease whose specificity matches that of the furin family of proprotein convertases. The expression of BAFF receptor appeared to be restricted to B cells. Both membrane-bound and soluble BAFF induced proliferation of anti-immunoglobulin M-stimulated peripheral blood B lymphocytes. Moreover, increased amounts of immunoglobulins were found in supernatants of germinal center-like B cells costimulated with BAFF. These results suggest that BAFF plays an important role as costimulator of B cell proliferation and function.

Long-term culture of human CD34(+) progenitors with FLT3-ligand, thrombopoietin, and stem cell factor induces extensive amplification of a CD34(-)CD14(-) and a CD34(-)CD14(+) dendritic cell precursor.

Current in vitro culture systems allow the generation of human dendritic cells (DCs), but the output of mature cells remains modest. This contrasts with the extensive amplification of hematopoietic progenitors achieved when culturing CD34(+) cells with FLT3-ligand and thrombopoietin. To test whether such cultures contained DC precursors, CD34(+) cord blood cells were incubated with the above cytokines, inducing on the mean a 250-fold and a 16,600-fold increase in total cell number after 4 and 8 weeks, respectively. The addition of stem cell factor induced a further fivefold increase in proliferation. The majority of the cells produced were CD34(-)CD1a- CD14(+) (p14(+)) and CD34(-)CD1a-CD14(-) (p14(-)) and did not display the morphology, surface markers, or allostimulatory capacity of DC. When cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), both subsets differentiated without further proliferation into immature (CD1a+, CD14(-), CD83(-)) macropinocytic DC. Mature (CD1a+, CD14(-), CD83(+)) DCs with high allostimulatory activity were generated if such cultures were supplemented with tumor necrosis factor-alpha (TNF). In addition, p14(-) cells generated CD14(+) cells with GM-CSF and TNF, which in turn, differentiated into DC when exposed to GM-CSF and IL-4. Similar results were obtained with frozen DC precursors and also when using pooled human serum AB+ instead of bovine serum, emphasizing that this system using CD34(+) cells may improve future prospects for immunotherapy.

Quiescent memory B cells in human peripheral blood co-express bcl-2 and bcl-x(L) anti-apoptotic proteins at high levels.

The anti-apoptotic proteins bcl-2 and bcl-xL seem to exhibit strictly opposite expression patterns in normal lymphoid cell differentiation stages, with bcl-2 low and bxl-xL high in immature and mature proliferating cells, the reverse being the case in recirculating quiescent cells. However, it is in fact not known whether recirculating memory cells are bcl-xL low or high. We analyzed memory (immunoglobulin isotype-switched) B cells in human peripheral blood, which were small lymphocytes in the G0 phase of the cell cycle, but proliferated better than naive B cells in response to Staphylococcus aureus Cowan I. Ex vivo these cells co-expressed bcl-2 together with bcl-xL mRNA and protein at high levels. The mcl-1 mRNA level was low. The bcl-xL mRNA level decreased during culture in medium containing fetal calf serum, which implies that it is maintained in vivo by continuous or frequent, non-mitogenic signal(s). The high bcl-xL expression of memory B cells may be relevant with regard to their longevity and/or their capacity to undergo an accelerated secondary type immune response.

Memory, but not naive, peripheral blood B lymphocytes differentiate into Ig-secreting cells after CD40 ligation and costimulation with IL-4 and the differentiation factors IL-2, IL-10, and IL-3.

The ligation of CD40 on B lymphocytes by CD40 ligand, transiently expressed on activated Th cells, provides a key activation signal required for the germinal center B cell response. In vitro, human B cell activation has been investigated extensively by coculturing tonsillar B cells with CD32-transfected fibroblasts coated with anti-CD40 Abs, in the presence of cytokines (the CD40 system). When tonsillar IgD+ B cells are cultured in the CD40 system with IL-4, cells proliferate and switch to IgG, but they display a block of differentiation illustrated by the persistence of IgD expression on cycling B cells. In this study, we analyzed the responses of peripheral blood B lymphocyte fractions, which may contain fewer in vivo activated cells than those from tonsils. While the differentiation block was confirmed with peripheral naive B cells cultured in the CD40 system with IL-4, it was also observed with the combination of IL-2, IL-10, and IL-3 alone or together with IL-4 (persistence of >90% IgD+ cells, including 24-60% IgD+, IgG+ cells, and <6% IgD+, IgA+ cells after 8 days). IgD+, IgG-, and IgA- (naive) B cells secreted 70-fold less Ig than IgG+, IgA+ (memory) B cells in response to anti-CD40 plus IL-2, IL-10, and IL-3. IgG-, IgA- B cells, or IgD-, IgM+, which should include IgM+ memory cells, strongly secreted IgM, but no IgG. In conclusion, only memory B cells secreted Ig; like memory T cells, their activation requirements to differentiate into effector cells seem less stringent than those of the naive cells.

Key differentiation steps in normal B cells and in myeloma cells.

The malignant plasma cell in multiple myeloma expresses a monoclonal immunoglobulin (Ig) with a somatically mutated variable region. In humans, somatic hypermutation of Ig variable regions only occurs in mature B cells, during the helper T-cell (TH)-dependent germinal center (GC) reaction. Within this context, the major differentiation steps in normal B cells will be discussed: B-cell maturation, B-cell activation, the GC reaction--during which the B cells strongly proliferate and somatic hypermutation in conjunction with stringent cell selection leads to antibody affinity maturation--and the differentiation of B cells into plasma cells or memory cells. The myeloma cell resembles a normal plasma cell with regard to many of its biologic features, such as its homing to the bone marrow, interaction with stromal cells, or even its capacity to suppress hematopoiesis. While the expression of interleukin-6 (IL-6) and IL-10 disappears during normal differentiation of B cells into plasma cells, that of transforming growth factor beta 1 persists. Thus, normal plasma cells in principle could, like myeloma cells, suppress hematopoiesis if their proportion in the bone marrow greatly increased (this could occur, for example, in bone marrow aplasia). The specific key alteration leading to multiple myeloma still remains to be identified.

Human naive B cells cultured with EL-4 T cells mimic a germinal center-related B cell stage before generating plasma cells. Concordant changes in Bcl-2 protein and messenger RNA levels.

The T cell-dependent B cell response in vivo occurs in organized microenvironments. Alternative routes exist in that early plasma cells are generated in the T zone while others emerge later from the germinal center (GC) reaction. We investigated whether B cell stages resembling those defined in vivo/ex vivo might be induced in an in vitro system in which naive human B cells are activated by EL-4 T cells and cytokines. Adult peripheral blood- or cord blood-derived B cells were found to mimic an early activated stage (CD38(low), IgD+, increased CD5+) followed by a centroblastic GC-related stage (CD38(int), CD77+, CD95(Fas)+, Bcl-2 protein(low)) before differentiating into morphologically typical, CD38(high), Fas- plasma cells of an immature type (Bcl-2(low), VLA-5-). The GC-related cells and the plasma cells exhibited spontaneous apoptosis in medium, the former also undergoing anti-Fas antibody-induced apoptosis in medium as well as during CD40L exposure in the EL-4 cultures. These Bcl-2(low) cells maintained a high viability in contact with EL-4 cells. Thus, some, major B cell stages with typical functional features as described for cells in vivo/ex vivo are sequentially generated in this in vitro system and the kinetics of the changes can be analyzed in a synchronized cell population. With regard to previous apparently conflicting observations on the Bcl-2 mRNA level in GC B cells, we performed competitive reverse-transcription polymerase chain reaction. Concordant changes in Bcl-2 mRNA and protein levels were found, i.e. during Bcl-2 down-regulation in the GC-related B cells in ongoing EL-4 cultures or in medium, and during a more modest up-regulation upon contact with fresh EL-4 cells. Regulation of Bcl-2 protein, therefore, predominantly occurred at the mRNA steady-state level.

Interleukin-2 promoter activity in Epstein-Barr virus-transformed B lymphocytes is controlled by nuclear factor-chi B.

The regulation of interleukin (IL)-2 gene expression has been investigated mainly in T lymphocytes, the predominant producers of IL-2. However, B cells can also synthesize IL-2. In the present study we analyzed the control of IL-2 promoter activity in Epstein-Barr virus (EBV)-transformed B cell clones which are capable of secreting IL-2 at a low level after stimulation with phorbol 12-myristate 13-acetate and the Ca2+ ionophore ionomycin. Transient transfections using reporter constructs with multiples of transcription factor binding sites from the IL-2 promoter [distal nuclear factor (NF)-AT, proximal NF-AT, AP-1/Octamer (UPS) or NF-chi B (TCEd) sites] were performed. In EBV-transformed B clones, the chi B site exerted the strongest inducible activity; the NF-AT binding sites showed either no or only weak activity compared to Jurkat T cells. An IL-2 promoter bearing a defective NF-chi B site was completely inactive in EBV-transformed B cells, while it still had activity in Jurkat T cells. In seven EBV-B cell clones or lines differing in their capacity to secrete IL-2, the activity of the IL-2 promoter correlated well with the status of IL-2 secretion. Similarly, a human immunodeficiency virus promoter, whose activity is controlled through chi B factors, was found to be active in the IL-2 producing EBV-B cells, but inactive in the non-IL-2-producing cells. Electrophoretic mobility shift assays using protein extracts from EBV-B cells and the IL-2 NF-chi B probe revealed the constitutive generation of chi B complexes in IL-2-secreting cells consisting mainly of heterodimeric p50/p65 complexes. A weaker chi B complex formation and faster-migrating complexes were detected in non-IL-2-secreting cells. These results demonstrate that the IL-2 NF-chi B site is indispensable for the activity of the IL-2 promoter in EBV-transformed B cells, whereas other transcription factors appear to be less important for IL-2 expression in these cells.

Interleukin-2 secretion by human B lymphocytes occurs as a late event and requires additional stimulation after CD40 cross-linking.

While Epstein-Barr virus (EBV)-immortalized B cell lines have been shown to secrete interleukin (IL)-2 after stimulation with either teleocidin or phorbol myristate acetate (PMA) and ionomycin, experimental conditions leading to IL-2 production by normal human B cells have not been reported. In the present study we investigated various B cell activating conditions, including--by analogy to EBV-immortalized B lymphocytes--stimulation of B cells that are already proliferating (in cultures with IL-4 and immobilized anti-CD40 monoclonal antibody; the anti-CD40 system). This approach showed that B lymphocytes secreted IL-2 in the culture medium, but only if they were first activated for more than 24 h in the anti-CD40 system before exposure to PMA plus ionomycin. The production rate of IL-2 by B lymphocytes reached a maximum after 6 days of priming in such cultures followed by 48 h of stimulation with PMA plus ionomycin, corresponding to 7% or 15% of that of fresh CD4+ T cells activated, respectively, with phytohemagglutinin plus PMA, or with PMA plus ionomycin for 48 h. This IL-2 production could not be attributed to T cell contamination nor to EBV-infected B cells according to flow cytometric and reverse transcriptase-polymerase chain reaction analysis of cultured B cells. Lower IL-2 expression (detected only as mRNA synthesis) was also induced in the cultured B lymphocytes after incubation with cross-linking anti-IgM antibodies instead of PMA plus ionomycin. The appearance of IL-13 mRNA, but not IL-4 mRNA, was detected under the same stimulation conditions as for IL-2 mRNA. These results show that the production of IL-2 by normal B lymphocytes occurs as a late event relative to their activation and proliferation, and is in this respect subject to regulation different to that found in T lymphocytes.

[Regulation of transcription of the interleukin-2 gene in B-lymphocytes]. / Régulation de la transcription du gène de l'interleukin-2 dans les lymphocytes B.

Since most B cell clones immortalized with EBV virus can be induced to produce interleukin-2, a typical T cell cytokine, we studied the role of different elements of the IL-2 promoter in such clones by transfection. It was found, in particular, that the element TCEd, which binds the transcription factor NF-kB, is very active in all three B clones tested. This element has no activity in T cells of the Jurkat line. The NFATd element, which binds the transcription factor NFAT-1 and is very active in T cells, is only weakly active in one B clone and not at all in another. Different elements thus contribute to IL-2 promoter activity in different cells.

Cytokine expression and regulation of human plasma cells: disappearance of interleukin-10 and persistence of transforming growth factor-beta 1.

Less is known about the cytokine expression and regulation of normal plasma cells compared to that of activated B cells or myeloma cells. This study shows that nonproliferating (hydroxyurea-treated), immunoglobulin (Ig)-secreting cells generated from human B cells in the EL-4 culture system no longer express interleukin (IL)-6 mRNA, progressively lose IL-10 mRNA, but continue to express transforming growth factor (TGF)-beta 1 mRNA. Secretion of TGF-beta 1 protein was demonstrated. On the other hand, and in contrast to the suppression of B cell proliferation and Ig secretion, the basal or the IL-6/IL-10 stimulated Ig secretion of nonproliferating cells was not inhibited by recombinant TGF-beta 1. Plasma cells isolated from human bone marrow expressed neither IL-6 nor IL-10 mRNA; only TGF-beta 1 mRNA was detected by reverse transcription-polymerase chain reaction analysis. Such plasma cells may be on average more "aged" cells than those generated in vitro. Thus, plasma cells persistently express TGF-beta 1, a known suppressor of various lymphoid and hemopoietic cell activities, but do not limit their own Ig secretion via this cytokine.

Transfusion-associated graft-versus-host disease in a patient treated with Cladribine (2-chlorodeoxyadenosine): demonstration of exogenous DNA in various tissue extracts by PCR analysis.

Transfusion-associated graft-versus-host disease can occur in both immunocompetent and immunocompromised hosts. Cladribine is a synthetic analogue of adenine used in the treatment of lymphoid malignancies, commonly associated with a decrease in T lymphocytes. Cladribine was given for a low-grade non-Hodgkin's lymphoma with thrombocytopenia as the main side-effect. Six units of pooled non-irradiated platelets were transfused from six unrelated donors; 10 d later a clinical picture typical of graft-versus-host disease resulted. Polymerase chain reaction of the highly polymorphic DNA minisatellites and HLA-DR oligotyping were used to demonstrate the exogenous DNA. In the patient's blood and tissues, only the pattern of donor 5 was found. The patient (DRB1*0301/1101; DRB3*0101/02) and this donor (DRB1*0301/1104; DRB3*02) by chance shared a partial common haplotype. This complication highlights the sensitivity of DNA minisatellite analysis. It further raises the question of transfusion and of prophylactic irradiation of all blood products in immunosuppressed patients and those treated with cladribine. This case represents a previously unreported situation where an immunosuppressed patient was able to eliminate cells from five totally HLA-DR dissimilar donors but not from one heterozygous donor with strong HLA-DR similarity.

Semiquantitative, nonradioactive RT-PCR detection of immunoglobulin mRNA in human B cells and plasma cells.

Quantification of mRNA is important for studies of gene expression and gene regulation. We investigated the utility of the reverse transcriptase polymerase chain reaction (RT-PCR) approach in the quantification of mRNA from small cell numbers. To take into account the complex kinetics of the PCR amplification process and the nonlinear signal development during detection of PCR products, calibration curves were established on the basis of different, known, starting concentrations of cDNA fragments, different PCR cycle numbers, and different signal intensities. Detection of digoxigenin-labeled PCR products via an enzymatically generated chemiluminescent signal was found to give a reproducible and wider range of signal intensities compared to simple ethidium bromide staining. We applied this methodology to the quantification of immunoglobulin M (IgM) mRNA levels in human B cells. Using an in vitro culture system in which B cells differentiate into plasma cells, the kinetics of IgM mRNA expression were established during a 10-day culture period and a 180-fold mRNA increase was found.